Fluorescent proteins are widely used in the fields of biochemistry, molecular and cell biology, medical diagnostics and drug screening methodologies (Chalfie et al., 1994, Science 263: 802-805; Tsien, 1998, Ann. Rev. Biochem. 67: 509-544). One property shared by the most useful fluorescent proteins is that they require no host-encoded co-factors or substrates for fluorescence. The proteins therefore retain their fluorescent properties both in isolation from their native organism, and when expressed in the cells of other organisms. This property makes them particularly well suited for a variety of in vivo and in vitro applications. Another major advantage of fluorescent proteins for use in biological systems is that they are indeed proteins, which permits their synthesis, within cells or organisms of interest, avoiding a host of problems relating to the attachment of the label to a protein of interest and/or delivery of labeled proteins into a cell. Not only can the proteins be made within the desired cell or organism, but they also retain their fluorescent properties when expressed as fusions with other proteins of interest, which greatly enhances their utility both in vivo and in vitro.
Fluorescent proteins have been used as reporter molecules to study gene expression in culture as well as in transgenic animals by insertion of fluorescent protein coding sequences downstream of an appropriate promoter. They have also been used to study the subcellular localization of proteins by direct fusion of test proteins to fluorescent proteins, and fluorescent proteins have become the reporter of choice for monitoring the infection efficiency of viral vectors both in cell culture and in animals. Variants of fluorescent proteins exhibiting spectral shifts in response to changes in the cellular environment (e.g., changes in pH, ion flux, or the redox status of the cell) are also used to monitor such changes (see, for example, Inouye & Tsuji, 1994, FEBS Lett. 351: 211-214; Miyawaki et al., 1997, Nature 388: 882-887).
Perhaps the most promising role for fluorescent proteins as biochemical markers is their application to methods that exploit fluorescence resonance energy transfer (FRET). FRET occurs with fluorophores for which the emission spectrum of one fluorophore overlaps with the excitation spectrum of a second fluorophore. When such fluorophores are brought into close proximity, excitation of the “donor” fluorophore results in emission from the “acceptor”. Pairs of such fluorophores are thus useful for monitoring molecular interactions. Fluorescent proteins are useful for the analysis of protein:protein molecular interactions in vivo or in vitro if their respective fluorescent emission and excitation spectra overlap to allow FRET. The donor and acceptor fluorescent proteins may be produced as fusions with the proteins one wishes to analyze for interactions. These types of applications of fluorescent proteins are particularly appealing for high throughput analyses, since the readout is direct and independent of subcellular localization.
The prototypical fluorescent protein is the Aequorea victoria green fluorescent protein (GFP), which was the first green fluorescent protein cloned (Prasher et al., 1992, Gene 111: 229-233). Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs blue light with an excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission wavelength of about 510 nm and a minor peak near 540 nm (Ward et al., 1979, Photochem. Photobiol. Rev. 4: 1-57). The polypeptide has several drawbacks, including relatively broad excitation and emission spectra, low quantum yield, and low expression in cells of higher eukaryotes. Mutants with improved spectral characteristics and higher quantum yield have been identified, and expression in higher eukaryotes has been improved by “humanizing” the nucleic acid sequences to encode codons optimized for human or mammalian expression.
Additional fluorescent proteins include, but are not limited to those expressed by Discosoma sp. and Phialidium gregarum (Ward et al., 1982, Photochem. Photobiol. 35: 803-808; Levine et al., 1982, Comp. Biochem. Physiol. 72B:77-85). Also, Vibrio fischeri strain Y1 expresses a yellow fluorescent protein that requires flavins as a co-factor for its fluorescence (Baldwin et al., 1990, Biochemistry 29: 5509-5515).
Additional cloned fluorescent proteins include, for example, the green fluorescent proteins from the sea pansy, Renilla mullerei (WO/99/49019) and from Renilla reniformis (see SEQ ID NO: 1; FIG. 1). Each of these fluorescent proteins and others are useful for a variety of in vivo and in vitro uses. The R. reniformis GFP (rGFP) clone is particularly important, since rGFP is seen as the benchmark protein among known naturally-occurring fluorescent proteins. rGFP has 3 to 6-fold higher quantum yield than A. victoria GFP, and the excitation and emission spectra are narrower, making rGFP more suitable for applications involving, for example, FRET.
One major drawback shared by the GFPs from A. victoria, R. mullerei and R. reniformis, as well as by all known variants of those proteins, is that they are dimeric. Generally, the proteins exist as homodimers. However, when more than one form of a given GFP is expressed in a single cell or is mixed in vitro, heterodimers can form if the dimerization interfaces for the different fluorescent proteins are complementary. Heterodimerization interferes with the usefulness of fluorescent proteins for several reasons.
First, heterodimerization is undesirable when fluorescent proteins are used in energy transfer-based analyses because heterodimerization raises the background of acceptor fluorescence without a real interaction between the proteins or protein domains of interest. When FRET is used, for example to monitor protein:protein interactions, donor and acceptor fluorescent fusion proteins are often expressed in the same cell or otherwise mixed. In the absence of heterodimerization, the excitation of the donor fluorophore leads to emission by the acceptor fluorophore only if the two fusion proteins are in close apposition. However, if heterodimerization occurs between the differing fluorescent proteins (e.g., between a wild-type rGFP and an rGFP variant that is a fluorescence donor to the wild-type GFP), excitation of the donor will result in emission by the acceptor regardless of the interaction between the fused polypeptides being examined for interaction. This generates an unacceptably high background fluorescence from the acceptor fluorophore.
Another problem caused by the heterodimerization is that the dimerization interfaces between the proteins can serve to artifactually bring fusion polypeptides linked to the fluorescent protein monomers into close contact. The inappropriate recruitment of proteins into close apposition can have biological consequences that make data interpretation difficult. For example, some cell surface receptors gain the ability to initiate an intracellular signaling cascade following ligand-induced dimerization. If the dimerization interfaces of the fluorescent proteins inappropriately recruit the fused receptor monomers into close contact, the signaling cascade can be inappropriately initiated in the absence of ligand. There is a need in the art for fluorescent proteins that do not heterodimerize.
U.S. Pat. No. 5,981,200 (Tsien et al.) teaches donor and acceptor fluorescent proteins linked by a peptide linker. The linked donor and acceptor proteins, referred to as “tandem fluorescent proteins,” are taught to be useful for assaying enzymes capable of cleaving the linker peptide sequence. When linked, the tandem fluorescent proteins exhibit either no fluorescence (e.g., when one protein quenches the fluorescence of the other) or fluorescence characteristic of the acceptor. Following cleavage, the fluorescence emitted is that characteristic of the individual fluorescent proteins. Assays using this arrangement will not work unless the tandem fluorescent proteins are related as donor and acceptor.